Comparison of two free light chain assays: performance of the involved free light chain ratio and implications for diagnosis of multiple myeloma

Dear Editor, The free light chain (FLC) assay is a valuable tool to screen and prognosticate plasma cell disorders (PCDs), follow response to therapy and progression. The de ﬁ nition of multiple myeloma (MM) requiring therapy was updated in 2014 by the International Myeloma Working Group (IMWG) to include the use of the FLC as an independent criterion for diagnosis of MM even in the absence of hypercalcemia, renal insuf ﬁ ciency, anemia or bone lesions (CRAB), and patients with myeloma de ﬁ ning events (MDE: more than one bone lesion by MRI; bone marrow plasmacytosis of greater than 60%; or an involved to uninvolved FLC ratio (iFLCr) of 100 or more along with an involved FLC concentration (iFLC) of at least 100 mg/L were recategorized as MM instead of SMM [1]. The original studies that prompted the change in SMM and MM de ﬁ nitions found the iFLCr cutoff of 100 or more to be associated with a 97% speci ﬁ city of progression to MM or AL at 2 years. All initial studies were done using the FreeLite TM reagents (The Binding Site, Birmingham, UK). Other manufacturers have recently developed their own FLC assays. Sebia developed an FLC assay, which like the FreeLite assay uses polyclonal antibodies to FLCs. Unlike the FreeLite assay, which depends on nephelometry or turbidimetry, the Sebia assay is an ELISA method amenable to automation in multiple platforms without a change in how the samples are processed. It is known that there is a less than ideal linear correlation between the tests [2 – 4] (and our data is shown as supplemental materials), but little is known about the performance of the iFLCr cut-points between tests. In this work, we focus on two important


Study Cohort
This study was reviewed and approved by the Mayo Clinic Institutional Review Board. Briefly, patients were identified by searching a computerized database and reviewing the medical records of patients who met the IMWG 2003 definition of SMM (1): >10% BMPCs and/or serum M-protein ≥30 g/L, plus absence of CRAB attributable to a PCD (calcium >0.25 mmol/L above the reference interval or > 2.75 mmol/L (>11.5 mg/dL), serum creatinine > 173 µmol/L (>2 mg/dL), hemoglobin 2 g/dL below the reference interval or <10 g/dL, or lytic lesions or diffuse osteopenia). From the original cohort of 586 of newly diagnosed SMM patients seen at the Mayo Clinic from 1976 to 2011 (2,3) with authorization to review their medical records for research purposes, we excluded (n=302) those patients: 1) with no remaining stored serum sample within 30 days of SMM diagnosis; or 2) who had received prior chemotherapy at the time of SMM diagnosis (4). Events documenting progression to a malignant PCD (MM or amyloidosis, AL) and death were captured in our myeloma database via chart review and annual follow-up letters to patients.

Serum FLC assays
Aliquots of frozen stored serum were retrieved, and Sebia FLC tests were performed by ELISA. The ELISAs were run on a DS2 (Dynex Technologies, Chantilly, VA) automated platform and the program provided by Sebia. Per protocol, the sample is diluted 1:1 000 and incubated in a microplate where anti-kappa (or anti-lambda) specific antibodies are fixed. After washing the excess non-fixed mixture, anti-kappa (or anti-lambda) antiserum conjugated to peroxidase is pipetted onto the plate. After addition of the peroxidase substrate, the reaction is stopped with acid and the optical density is read by absorbance spectrophotometry at 450nm. The DS2 instrument can perform one plate at a time and the analytical run of one 96-well plate for kappa (or lambda) FLC takes between 2.5-3h. FreeLite serum FLC results were from the medical record or research database from the 2008/2013 studies. Briefly, FreeLite reagents use polyclonal sheep-antihuman antibodies to FLC, and testing was performed on Siemens BN2 (Siemens, Marburg, Germany) nephelometric analyzers. Reported values for both assays include kappa and lambda concentration along with a kappa/lambda ratio. For this study, the calculation of the iFLCr was done according to the isotype identified on immunofixation electrophoresis or the FreeLite serum FLC assay.

Other laboratory tests
As part of the patients' routine care, other laboratory analyses were ordered by the providers and information recorded in the myeloma research database. Information was collected for serum protein electrophoresis (Helena Laboratories, Belmont, TX), serum immunofixation (Sebia Inc) and BMPC measured on biopsy, when available within 30 days of the SMM diagnosis. Hemoglobin was retrieved from the complete blood count (CBC), creatinine and serum calcium from the central clinical laboratory tests at the time of the blood collection. Estimated glomerular filtration rate (eGFR) was calculated with the CKD-EPI equation using the standardized creatinine value (5, 6).

Statistical analysis
All patients were included for the population demographics. Data is summarized as median and range for continuous data and number and percent for categorical variables. Assays were compared using Passing-Bablok linear regression analysis for FLC measures. Estimates of sensitivity and specificity of the assays were compared at established cut-points for the FLC assays.
Progression was defined as a new clinical diagnosis of MM or AL after the SMM diagnosis. Time to progression was calculated based on MM or AL diagnosis date. Patients who were considered SMM in 2003 but who would have met 2014 criteria of MM requiring therapy (7) were excluded (n=46) for the analysis of the 20-20-20 system for SMM stratification. Cox proportional hazards models, adjusting for death as a competing risk, were used to assess univariate survival -measured as time to any progression from SMM diagnosis, as well as to evaluate the 20-20-20 system. The c-statistic was used to assess the model's ability to predict any progression of SMM.

Brief analytical comparison between FreeLite and Sebia FLC assays
Serum free light chain assays by The Binding Site (FreeLite) and Sebia were compared. FreeLite kappa was missing for 20 patients, FreeLite lambda was missing for 20 patients, and there was overlap of missing data in both for 19 patients. Passing-Bablok linear regression is shown for kappa, lambda, kappa/lambda ratio and the iFLCr (Supplemental Figure 1). Slopes ranged from 0.634 for iFLCr to 0.747 for the kappa/lambda ratio.
Overall, when patients were categorized based on whether they had kappa or lambda restricted disease, the median involved FLC concentration and iFLCr were similar between FreeLite and Sebia, but due to the different analytical measurable ranges between assays, several of the comparisons between tests were statistically significant. In general, using the FreeLite assay, the values ran higher. The Sebia assay had a narrower range for kappa FLC and kappa iFLCr compared to FreeLite. In contrast, lambda FLC range was wider with Sebia (Table 1, main manuscript content).

Discussion of findings
Having multiple assays to measure FLC available on the market will ultimately improve the diagnosis and monitoring of PCDs, enabling smaller laboratories to offer the testing with ease. The disadvantage that may arise, as it happened with a few other laboratory analytes such as hemoglobin A1C (8-10) and prothrombin time (11), is the lack of harmonization between assays, which can lead to confusion and poor commutability of results across institutions. Supplemental Table 2 demonstrates the different FLC assays on the market and their differences. A common question that clinical laboratories receive when reevaluating their FLC assay and considering a move to a different manufacturer is if the clinical decision points for these new reagents would be equivalent to the FreeLite assay. Our study suggests that it is acceptable to apply the existing FLC criteria used to define MM requiring therapy and to predict SMM risk of progression when using the Sebia assay.
In Scandinavia, Siemens has the largest market share of instruments in clinical laboratories, and groups using those reagents reported that the finding of an iFLC concentration of 100 mg/L or more could be applied to their N-Latex FLC assays as a risk factor for MGUS progression to MM (12,13). The Sebia FLC ELISA method faces similar challenges as one of the newest reagents to reach clinical laboratories. Here we show that the iFLCr of 100 or more can be used for Sebia FLC assays with a sensitivity of 16.7% and specificity of 93.1%, virtually identical to FreeLite reagents, using a subset of the patients' samples originally used to define the cutpoints in the IMWG 2014 guideline.
In a previous study with the Sebia FLC assay, newly diagnosed MM patients (177 patients with 368 samples) were tested simultaneously with FreeLite and Sebia FLC reagents (14). Authors verified the modest correlation between the methods that we also observed in this study and found a Pearson correlation coefficient r=0.713 for the iFLCr in diagnostics samples. Using a mathematical linear regression they concluded that the FreeLite cut-point of 100 could be converted to a cut-point of 16 using Sebia FLC (14).
Another study compared 47 patients (31 newly diagnosed MM cases and 16 relapsed/refractory MM), with 177 samples (15). This group also verified the modest correlation between the assays when quantitatively comparing the individual concentrations of kappa and lambda or the iFLCr (15). To find comparable cut-points between the tests they used Cohen's Kappa coefficients and concluded The Sebia FLC cut-point of 100 was roughly equivalent to 20 using Sebia reagents, and the 20-20-20 system cut-point should be replaced for iFLCr of 8 using Sebia reagents (15). These two studies used a heterogenous group of active MM patients, and not a cohort of SMM to examine the iFLCr ≥ 100 cut-point. The findings from previous studies are difficult to compare to our current findings, as the study design from those is different from ours and from the original studies that defined the iFLCr ≥ 100 (4, 7). The use of conversion factors, while seeming practical for rebaselining subjects after a test change, are not the most appropriate tool to use in the clinical laboratory when the measurements are highly skewed and do not follow a Gaussian distribution. FreeLite and Sebia assays have a correlation coefficient (r) not exceeding 0.9, which means the assays show wide variance and results are not linearly correlated, especially for high concentrations of FLC, so often the conversion factor utilized may not represent the actual experimental test result.
Considering the use of the 20-20-20 system for high-risk SMM, the study shows that performance of the Sebia ELISA is at least as good as FreeLite, Supplemental Table 3.
In conclusion, despite the analytical differences between assays, we were able to show that the diagnostic value of the iFLCr ≥100 for patients. This commutability of the iFLCr cut-points in the SMM cohort is welcome in the hematology field and provides significant information to clinicians first seeing these patients and categorizing them as having a pre-malignant or malignant PCD. Tables   Supplemental table 1